Summary

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Published: July 02, 2015
doi:

Summary

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Abstract

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 µm because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

Introduction

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 × 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.

Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg−Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents.

Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

Protocol

1. Vorbereitung Materialien Herstellung von 10 mM HEPES-Puffer. Den pH-Wert auf 7,0 bei RT und fügen NaCl auf 0,9%. Bereiten Sie 5% Alginat-Lösung und 10 mM EDTA-Lösung durch Mischen von HEPES-gepufferte Kochsalzlösung in 1.1 vorbereitet. Man stellt den pH auf 7,0 bei RT. Autoklaven werden die Reagenzien (1.1, 1.2) für 20 Minuten bei 121 ° C. Vorbereitung Gelatine beschichteten 60 mm-Schalen mit 1,2 geschichtet – 2,0 × 10 6 embryonalen Maus-Fibroblasten (MEF) Ze…

Representative Results

Bei Protokoll 2.5, die vertrieben Alginat-Lösung bildet sofort eine Kugelform nach Vertreibung (2A – H). Wenn die Suspension mit einer N 2 Durchflussrate von weniger als 1 l / min ausgestoßen, der Größe der Tröpfchen einheitlicher (2I). Wenn die N 2 -Strom größer ist als 1 l / min, unterbricht jedoch die Tröpfchen unten (Figur 2G, weiße Pfeile) und die Größe der Tröpfchen heterogen (2J). Angesichts dieser Tatsache ist e…

Discussion

Encapsulation Kultur kann mit direktem Suspensionskulturen verglichen werden. Suspensionskultur ist eine einfachere Methode, um große Mengen von pluripotenten Stammzellen als Einkapselungsverfahren erhalten. , Steuerung der Aggregation von Zellen in Suspensionskultur ist jedoch immer noch eine Herausforderung. Im Verkapselungsverfahren wird Zellaggregation in Kapseln beschränkt und kann daher gut kontrolliert werden. In einer früheren Publikation zeigte, dass eingekapselten Zellen gebildeten Aggregate von einhe…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

Materials

Mouse embryo fibroblast Cell Biolabs SNL 76/7
Mouse induced pluripotent stem cell RIKEN Bio resorce centre iPS-MEF-Ng-20D-17
DMEM high-glucose GIBCO 11995
ES qualified  FBS GIBCO 16141079
Antibacterial Antibiotics GIBCO 15240
Nonessential Amino Acid GIBCO 11140
2-mercaptoethanol GIBCO 21985-023
ESGRO Leukemia Inhibitory Factor Merck Millipore ESG1107
Trypsin/EDTA GIBCO 25300
26G/16G needle Hoshiseido
10 mL Syringe TERUMO SS-10ESZ
Sodium  Chloride Wako 191-01665
HEPES SIGMA H4034
Sodium Alginate Wako 194-09955
Calcium Chloride Wako 039-00475
Poly-L-lysine (Mw=15,000-30,000) SIGMA P7890
EDTA DOJINDO 345-01865
Sylinge pump AS ONE
Microscope Olympus IX71
Microscope Leica DM IRB
Hispeed camera nac image technology Memrecam HX-3

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Cite This Article
Horiguchi, I., Sakai, Y. Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle. J. Vis. Exp. (101), e52835, doi:10.3791/52835 (2015).

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